Enterohemorrhagic escherichia coli vaccine

ABSTRACT

Compositions and methods for stimulating an immune response against a secreted enterohemorragic  Escherichia coli  (EHEC) antigen are disclosed. The compositions comprise EHEC cell culture supernatants.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 USC §119(e)(1) ofprovisional patent application serial no. 60/259,818, filed Jan. 4,2001, which application is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods foreliciting an immune response in mammals against enterohemorragicEscherichia coli. In particular, the invention relates to the use ofcell culture supernatants for treating and preventing enterohemorragicE. coli colonization of mammals.

BACKGROUND OF THE INVENTION

[0003] Enterohemorragic Escherichia coli (EHEC), also called Shiga toxinE. coli (STEC) and vertotoxigenic E. coli (VTEC) are pathogenic bacteriathat cause diarrhea, hemorrhagic colitis, hemolytic uremic syndrome,kidney failure and death in humans. While many Shiga-liketoxin-producing EHEC strains are capable of causing disease in humans,those of serotype O157:H7 cause the majority of human illness. Thisorganism is able to colonize the large intestine of humans by a uniquemechanism in which a number of virulence determinants are delivered tohost cells via a type III secretion system, including the translocatedIntimin receptor, Tir (DeVinney et al., Infect. Immun. (1999) 67:2389).In particular, these pathogens secrete virulence determinants EspA, EspBand EspD that enable delivery of Tir into intestinal cell membranes. Tiris integrated into the host cell membrane where it serves as thereceptor for a bacterial outer membrane protein, Intimin. Tir-Intiminbinding attaches EHEC to the intestinal cell surface and triggers actincytoskeletal rearrangements beneath adherent EHEC that results inpedestal formation. EspA, EspB, Tir and Intimin are each essential forthe successful colonization of the intestine by EHEC.

[0004] Although EHEC colonize the intestine of ruminants and othermammals, they generally do not cause overt disease in these animals.However, contamination of meat and water by the EHEC serotype O157:H7(hereinafter, “EHEC O157:H7”) is responsible for about 50,000 cases ofEHEC O157:H7 infection in humans annually in the United States andCanada that result in approximately 500 deaths. In 1994, the economiccost associated with EHEC O157:H7 infection in humans was estimated tobe over 5 billion dollars annually.

[0005] The first documented EHEC O157:H7 outbreak traced to contaminatedmeat occurred in 1982. Subsequently, it was demonstrated that healthyruminants including, but not limited to, cattle, dairy cows and sheep,could be infected with EHEC O157:H7. In fact, USDA reports indicate thatup to 50% of cattle are carriers of EHEC O157:H7 at some time duringtheir lifetime and, therefore, shed EHEC O157:H7 in their feces.

[0006] Because of the bulk processing of slaughtered cattle and the lownumber of EHEC O157:H7 (10-100) necessary to infect a human, EHECO157:H7 colonization of healthy cattle remains a serious health problem.To address this problem, research has focused on improved methods fordetecting and subsequently killing EHEC O157:H7 at slaughter, alteringthe diet of cattle to reduce the number of intestinal EHEC O157:H7 andimmunizing animals to prevent EHEC O157:H7 colonization (Zacek D. AnimalHealth and Veterinary Vaccines, Alberta Research Counsel, Edmonton,Canada, 1997). Recently, the recombinant production and use of EHECO157:H7 proteins including recombinant EspA (International PublicationNo. WO 97/40063), recombinant TIR (International Publication No. WO99/24576), recombinant EspB and recombinant Initimin (Li et al., Infec.Immun. (2000) 68:5090-5095) have been described. However, production andpurification of recombinant proteins in amounts sufficient for use asantigens is both difficult and expensive. At the present time, there isno effective method for blocking EHEC O157:H7 colonization of cattle andother mammals and, thereby, for reducing shedding of EHEC into theenvironment.

[0007] Therefore, there is a need for new compositions and methods fortreating and preventing EHEC disease, as well as for reducing EHECcolonization of mammals in order to reduce the incidence of healthproblems associated with EHEC-contaminated meat and water.

SUMMARY OF THE INVENTION

[0008] The present invention satisfies the above need by providing suchcompositions and methods. In particular, the methods of the presentinvention make use of a composition comprising a cell culturesupernatant (hereinafter “CCS”) derived from an EHEC culture to elicitan immune response against one or more EHEC secreted antigens, therebytreating and/or preventing EHEC infection and/or reducing EHECcolonization of the mammal. The compositions can be delivered with orwithout a coadministered adjuvant. In certain embodiments, EspA and Tircomprise at least 20% of the cell culture supernatant protein. The EHECculture supernatant may be derived from any EHEC serotype, but ispreferably obtained from a culture of EHEC O157:H7 and/or EHEC O157:NM(non-motile). The cell culture supernatant of the present invention iseasy and relatively inexpensive to prepare and is effective at doseregimens that have minimal toxicity.

[0009] EspA, EspB, Tir and Intimin are necessary for activation (A) ofhost epithelial cell signal transduction pathways and for the intimateattachment (E) of EHEC to host epithelial cells. Therefore, withoutbeing bound by the following hypothesis, it is thought thatadministration of the CCS of the present invention to a mammalstimulates an immune response against one or more secreted antigens,such as EspA and Tir, that blocks attachment of the EHEC to intestinalepithelial cells.

[0010] Accordingly, it is an object of the present invention to providea vaccine effective to stimulate an immune response against EHECsecreted antigens, thereby treating and/or preventing EHEC disease in amammal.

[0011] Another object is to provide a vaccine effective to reduce,prevent and/or eliminate EHEC colonization of a ruminant or othermammal.

[0012] Another object is to reduce the number of animals shedding EHECinto the environment.

[0013] Another object is to reduce the number of EHEC shed into theenvironment by an infected animal.

[0014] Another object is reduce the time during which EHEC are shed intothe environment by an infected animal.

[0015] Another object is reduce EHEC contamination of the environment.

[0016] Another object is reduce EHEC contamination of meat and/or water.

[0017] Another object is to treat, prevent and/or reduce EHEC infectionsin humans.

[0018] Another object is to provide a vaccine effective as an adjunct toother biological anti-EHEC agents.

[0019] Another object is to provide a vaccine effective as an adjunct tochemical anti-EHEC agents.

[0020] Another object is to provide a vaccine effective as an adjunct tobiologically engineered anti-EHEC agents.

[0021] Another object is to provide a vaccine effective as an adjunct tonucleic acid-based anti-EHEC agents.

[0022] Another object is to provide a vaccine effective as an adjunct torecombinant protein anti-EHEC agents.

[0023] Another object is to provide a vaccination schedule effective toreduce EHEC colonization of a ruminant.

[0024] Another object is to provide a vaccination schedule effective toreduce EHEC shedding by a ruminant.

[0025] Another object is to provide a vaccine effective to reduce EHECO157 colonization of cattle, such as colonization of EHEC O157:H7 and/orEHEC O157:NM.

[0026] Another object is to provide a vaccine effective to prevent EHECO157 colonization of cattle, such as colonization of EHEC O157:H7 and/orEHEC O157:NM.

[0027] Another object is to provide a vaccine effective to eliminateEHEC O157 colonization of cattle, such as colonization of EHEC O157:H7and/or EHEC O157:NM.

[0028] Another object is to reduce the number of cattle shedding EHECO157 into the environment, such as shedding of EHEC O157:H7 and/or EHECO157:NM.

[0029] Another object is to reduce the number of EHEC O157 shed into theenvironment by infected cattle, such as shedding of EHEC O157:H7 and/orEHEC O157:NM.

[0030] Another object is reduce the time during which EHEC O157 are shedinto the environment by infected cattle, such as shedding of EHECO157:H7 and/or EHEC O157:NM.

[0031] Another object is to provide a vaccine effective as an adjunct toother anti-EHEC O157 agents.

[0032] Another object is to provide a vaccination schedule effective toreduce EHEC O157 colonization of cattle.

[0033] Another object is to provide a vaccination schedule effective toreduce EHEC O157 shedding by cattle.

[0034] Thus, in one embodiment, the invention is directed to a vaccinecomposition comprising an enterohemorragic Escherichia coli (EHEC) cellculture supernatant and an immunological adjuvant. In certainembodiments, the EHEC is EHEC O157:H7 and/or EHEC O157:NM. In additionalembodiments, the immunological adjuvant comprises an oil-in-wateremulsion, such as a mineral oil and dimethyldioctadecylammonium bromide.In yet additional embodiments, the immunological adjuvant is VSA3. TheVSA3 may be present at a concentration of about 20% to about 40% (v/v),such as at a concentration of 30% (v/v).

[0035] In still further embodiments, the vaccine composition furthercomprises one or more recombinant or purified EHEC secreted antigensselected from the group consisting of EspA, EspB, EspD and Tir. In otherembodiments, EspA+Tir comprise at least 20% of the cell protein presentin the composition.

[0036] In further embodiments, the subject invention is directed tomethods for eliciting an immunological response in a mammal against asecreted enterohemorragic Escherichia coli (EHEC) antigen. The methodcomprises administering to the mammal a therapeutically effective amountof a composition comprising an EHEC cell culture supernatant. In certainembodiments, the EHEC is EHEC O157:H7 and/or EHEC O157:NM. In additionalembodiments, the mammal is a human or a ruminant, such as a bovinesubject. In yet further embodiments, the composition further comprisesan immunological adjuvant, such as an oil-in-water emulsion whichcomprises e.g., a mineral oil and dimethyldioctadecylammonium bromide.In additional embodiments, the adjuvant is VSA3. The compositions mayfurther comprise one or more recombinant or purified EHEC secretedantigens selected from the group consisting of EspA, EspB, EspD and Tir.In other embodiments, EspA+Tir comprise at least 20% of the cell proteinpresent in the composition.

[0037] In another embodiment, the invention is directed to a method foreliciting an immunological response in a ruminant against a secretedenterohemorragic Escherichia coli O157:H7 (EHEC O157:H7) antigen. Themethod comprises administering to the ruminant a therapeuticallyeffective amount of a composition comprising an EHEC O157:H7 cellculture supernatant and VSA3. In additional embodiments, VSA3 is presentin the composition at a concentration of about 20% to about 40% (v/v),such as at about 30% (v/v).

[0038] In still a further embodiment, the invention is directed to amethod for reducing colonization of enterohemorragic Escherichia coli(EHEC) in a ruminant comprising administering to the ruminant atherapeutically effective amount of a composition comprising an EHECcell culture supernatant and an immunological adjuvant.

[0039] In yet another embodiment, the invention is directed to a methodfor reducing shedding of enterohemorragic Escherichia coli (EHEC) from aruminant comprising administering to the ruminant a therapeuticallyeffective amount of a composition comprising an EHEC cell culturesupernatant and an immunological adjuvant.

[0040] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings. In addition, various references are set forth hereinwhich describe in more detail certain procedures or compositions, andare therefore incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 shows the electrophoretic profile of CCS proteins separatedby polyacrylamide gel electrophoresis.

[0042]FIG. 2 shows the electrophoretic profile of recombinant EspA, Tir,EspB and Intimin separated by polyacrylamide gel electrophoresis.

[0043]FIG. 3 shows fecal shedding of EHEC O157:H7 by cattle immunizedwith a CCS vaccine following EHEC O157:H7 challenge.

[0044]FIG. 4 depicts reactivation of fecal shedding of EHEC O157:H7 inpreviously infected cattle.

[0045]FIG. 5 shows the serological response to immunization withrecombinant EspA+Tir vaccine and with recombinant EspB+Intimin vaccine.

[0046]FIG. 6 depicts fecal shedding of EHEC O157:H7 followingimmunization with recombinant EspA+Tir vaccine and with saline vaccine.

[0047]FIG. 7 shows the number of animals shedding E. coli O157:H7 oneach day of the vaccine trial described in Example 6. Bacteria weredetected by direct plating of fecal samples which had been resuspendedin saline on Sorbitol MaConkey agar supplemented with cefixime andtellurite. Solid bars, placebo group; hatched bars, EHEC vaccine group.

[0048]FIG. 8 shows an immunoblot analysis of sera from vaccinatedanimals against EHEC secreted proteins. Each blot contains secretedproteins from wild-type E. coli O157:H7 (EHEC), type III secretionmutant (ΔSepB), tir mutant (ΔTir) and a purifiedglutathione-s-transferase:Tir fusion protein (GST-Tir). Proteins wereseparated by SDS-10% PAGE and stained with Coomassie blue (A, upper leftpanel) or transferred to nitrocellulose and probed with representativesera from animals which received 3 immunizations with each vaccineformulation (A, upper panels). The lower four panels (B) were probedwith sera from one representative animal which received the EHECvaccine, taken on days 0, 21, 25 and 49 of the trial.

[0049]FIG. 9 shows the percentage of each group of animals shedding E.coli O157:H7(Panel A) and the total number of bacteria recovered (PanelB) on each day of the trial described in Example 6. Bacteria weredetected in feces by plating on Sorbitol MaConkey agar supplemented withcefixime and tellurite following immunomagnetic enrichment as describedin J. Van Donkersgoed et al., Can. Vet. J. (2001) 42:714. (A) Solidbars, placebo; hatched bars, EHEC vaccine; open bars, ΔTir vaccine. (B)▪, placebo group; , EHEC vaccine; ▴, ΔTir vaccine.

DETAILED DESCRIPTION OF THE INVENTION

[0050] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, recombinant DNA technology, and immunology, which arewithin the skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Vols. I, II and III, Second Edition (1989); Perbal,B., A Practical Guide to Molecular Cloning (1984); the series, MethodsIn Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C.C. Blackwell eds., 1986, Blackwell Scientific Publications).

[0051] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0052] A. Definitions

[0053] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0054] It must be noted that, as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “an EHEC bacterium ” includes a mixture of two ormore such bacteria, and the like.

[0055] As used herein, the term EHEC “cell culture supernatant” or “CCS”refers to a supernatant derived from a cell culture of one or more EHECserotypes, which supernatant is substantially free of EHEC bacterialcells or the lysate of such cells, and which contains a mixture of EHECantigens that have been secreted into the growth media. Generally, anEHEC “CCS” will contain at least the secreted antigens EspA, EspB, EspDand Tir, and fragments or aggregates thereof. The CCS of the presentinvention may also include other secreted proteins, such as EspF andMAP, one or both of Shiga toxins 1 and 2, as well as EspP which is anapproximately 100 kDa protein which is not secreted by the type IIIsystem. The proteins can be present in a native form, or a denatured ordegraded form, so long as the CCS still functions to stimulate an immuneresponse in the host subject such that EHEC disease is lessened orprevented, and/or colonization of EHEC is lessened or suppressed. Insome instances, a CCS may be supplemented with additional recombinant orpurified secreted antigens, such as with additional EspA, EspB, EspDand/or Tir, as well as with any of the other secreted proteins, and mayalso be supplemented with Intimin. In certain embodiments, EspA+Tir willcomprise at least 20% of the cell culture supernatant protein.

[0056] As used herein, a “recombinant” EHEC secreted protein, such asrEspA, rEspB, rEspD and rTir, as well as the “recombinant Intimin”,refers to the full-length polypeptide sequence, fragments of thereference sequence or substitutions, deletions and/or additions to thereference sequence, so long as the proteins retain at least one specificepitope or activity. Generally, analogs of the reference sequence willdisplay at least about 50% sequence identity, preferably at least about75% to 85% sequence identity, and even more preferably about 90% to 95%or more sequence identity, to the full-length reference sequence. See,e.g., GenBank Accession Nos. AE005594, AE005595, AP002566, AE005174,NC_(—)002695, NC_(—)002655 for the complete sequence of the E. coliO157:H7 genome, which includes the sequences of the various O157:H7secreted proteins. See, e.g., International Publication No. WO 97/40063,as well as GenBank Accession Nos. Y13068, U80908, U5681, Z54352,AJ225021, AJ225020, AJ225019, AJ225018, AJ225017, AJ225016, AJ225015,AF022236 and AF200363 for the nucleotide and amino acid sequences ofEspA from a number of E. coli serotypes. See, e.g., InternationalPublication No. WO 99/24576, as well as GenBank Accession Nos. AF125993,AF132728, AF045568, AF022236, AF70067, AF070068, AF013122, AF200363,AF113597, AF070069, AB036053, AB026719, U5904 and U59502, for thenucleotide and amino acid sequences of Tir from a number of E. coliserotypes. See, e.g., GenBank Accession Nos. U32312, U38618, U59503,U66102, AF081183, AF081182, AF130315, AF339751, AJ308551, AF301015,AF329681, AF319597, AJ275089-AJ 275113 for the nucleotide and amino acidsequences of Intimin from a number of E. coli serotypes. See, e.g.,GenBank Accession Nos. U80796, U65681, Y13068, Y13859, X96953, X99670,X96953, Z21555, AF254454, AF254455, AF254456, AF254457, AF054421,AF059713, AF144008, AF144009 for the nucleotide and amino acid sequencesof EspB from a number of E. coli serotypes. See, e.g., GenBank AccessionNos. Y13068, Y13859, Y17875, Y17874, Y09228, U65681, AF054421 andAF064683, for the nucleotide and amino acid sequences of EspD from anumber of E. coli serotypes.

[0057] “Homology” refers to the percent similarity between twopolynucleotide or two polypeptide moieties. Two DNA, or two polypeptidesequences are “substantially homologous” to each other when thesequences exhibit at least about 80%-85%, preferably at least about 90%,and most preferably at least about 95%-98% sequence similarity over adefined length of the molecules. As used herein, substantiallyhomologous also refers to sequences showing complete identity to thespecified DNA or polypeptide sequence.

[0058] Percent sequence identity can be determined by a directcomparison of the sequence information between two molecules by aligningthe sequences, counting the exact number of matches between the twoaligned sequences, dividing by the length of the shorter sequence, andmultiplying the result by 100. Readily available computer programs canbe used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlasof Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358,National biomedical Research Foundation, Washington, DC, which adaptsthe local homology algorithm of Smith and Waterman (1981) Advances inAppl. Math. 2:482-489 for peptide analysis. Programs for determiningnucleotide sequence identity are available in the Wisconsin SequenceAnalysis Package, Version 8 (available from Genetics Computer Group,Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, whichalso rely on the Smith and Waterman algorithm. These programs arereadily utilized with the default parameters recommended by themanufacturer and described in the Wisconsin Sequence Analysis Packagereferred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

[0059] Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

[0060] As used herein, “vaccine” refers to a CCS composition that servesto stimulate an immune response to an EHEC antigen, such as a type IIIsecreted EHEC antigen, therein. The immune response need not providecomplete protection and/or treatment against EHEC infection or againstcolonization and shedding of EHEC. Even partial protection againstcolonization and shedding of EHEC bacteria will find use herein asshedding and contaminated meat production will still be reduced. In somecases, a vaccine will include an immunological adjuvant in order toenhance the immune response. The term “adjuvant” refers to an agentwhich acts in a nonspecific manner to increase an immune response to aparticular antigen or combination of antigens, thus reducing thequantity of antigen necessary in any given vaccine, and/or the frequencyof injection necessary in order to generate an adequate immune responseto the antigen of interest. See, e.g., A. C. Allison J.Reticuloendothel. Soc. (1979) 26:619-630. Such adjuvants are describedfurther below.

[0061] As used herein, “colonization” refers to the presence of EHEC inthe intestinal tract of a mammal, such as a ruminant.

[0062] As used herein, “shedding” refers to the presence of EHEC infeces.

[0063] As used herein, “therapeutic amount”, “effective amount” and“amount effective to” refer to an amount of vaccine effective to elicitan immune response against a secreted antigen present in the CCS,thereby reducing or preventing EHEC disease, and/or EHEC colonization ofa mammal such as a ruminant; and/or reducing the number of animalsshedding EHEC; and/or reducing the number of EHEC shed by an animal;and/or, reducing the time period of EHEC shedding by an animal.

[0064] As used herein, “immunization” or “immunize” refers toadministration of CCS, with or without additional recombinant orpurified EHEC antigens such as EspA, Tir, EspB, EspD, and/or Intimin, inan amount effective to stimulate the immune system of the animal towhich the CCS is administered, to elicit an immunological responseagainst one or more of the secreted antigens present in the CCS.

[0065] The term “epitope” refers to the site on an antigen or hapten towhich specific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite.”

[0066] An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to the composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells and/or γδ T cells,directed specifically to an antigen or antigens included in thecomposition or vaccine of interest. Preferably, the host will displayeither a therapeutic or protective immunological response such that EHECdisease is lessened and/or prevented; resistance of the intestine tocolonization with EHEC is imparted; the number of animals shedding EHECis reduced; the number of EHEC shed by an animal is reduced; and/or thetime period of EHEC shedding by an animal is reduced.

[0067] The terms “immunogenic” protein or polypeptide refer to an aminoacid sequence which elicits an immunological response as describedabove. An “immunogenic” protein or polypeptide, as used herein, includesthe full-length sequence of the particular EHEC protein in question,analogs thereof, aggregates, or immunogenic fragments thereof. By“immunogenic fragment” is meant a fragment of a secreted EHEC proteinwhich includes one or more epitopes and thus elicits the immunologicalresponse described above. Such fragments can be identified using anynumber of epitope mapping techniques, well known in the art. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66(Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example,linear epitopes may be determined by e.g., concurrently synthesizinglarge numbers of peptides on solid supports, the peptides correspondingto portions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002;Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated hereinby reference in their entireties. Similarly, conformational epitopes arereadily identified by determining spatial conformation of amino acidssuch as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, supra.Antigenic regions of proteins can also be identified using standardantigenicity and hydropathy plots, such as those calculated using, e.g.,the Omiga version 1.0 software program available from the OxfordMolecular Group. This computer program employs the Hopp/Woods method,Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 fordetermining antigenicity profiles, and the Kyte-Doolittle technique,Kyte et al., J. Mol. Biol. (1982) 157:105-132 for hydropathy plots.

[0068] Immunogenic fragments, for purposes of the present invention,will usually include at least about 3 amino acids, preferably at leastabout 5 amino acids, more preferably at least about 10-15 amino acids,and most preferably 25 or more amino acids, of the parent EHEC secretedprotein molecule. There is no critical upper limit to the length of thefragment, which may comprise nearly the full-length of the proteinsequence, or even a fusion protein comprising two or more epitopes ofthe particular EHEC secreted protein.

[0069] “Native” proteins or polypeptides refer to proteins orpolypeptides isolated from the source in which the proteins naturallyoccur. “Recombinant” polypeptides refer to polypeptides produced byrecombinant DNA techniques; i.e., produced from cells transformed by anexogenous DNA construct encoding the desired polypeptide. “Synthetic”polypeptides are those prepared by chemical synthesis.

[0070] The term “treatment” as used herein refers to either (i) theprevention of infection or reinfection (prophylaxis), or (ii) thereduction or elimination of symptoms of the disease of interest(therapy).

[0071] By “mammalian subject” is meant any member of the class Mammalia,including humans and all other mammary gland possessing animals (bothmale and female), such as ruminants, including, but not limited to,bovine, porcine and Ovis (sheep and goats) species. The term does notdenote a particular age. Thus, adults, newborns, and fetuses areintended to be covered.

[0072] B. General Methods

[0073] Central to the present invention is the discovery that cellculture supernatants derived from EHEC cultures which contain EHECsecreted antigens, produce an immune response in animals to which theyare administered and thereby provide protection against EHEC infection,such as protection against colonization. In certain embodiments, thecompositions comprise a mixture of EHEC secreted antigens, including butnot limited to EspA, EspB, EspD and/or Tir. The CCS of the presentinvention may also include other secreted proteins, such as EspF andMAP, one or both of Shiga toxins 1 and 2, as well as EspP which is anapproximately 100 kDa protein which is not secreted by the type IIIsystem. In other embodiments, the CCS is supplemented with additionalrecombinant or purified EHEC antigens, such as with additional EspA,EspB, EspD, Tir, Intimin, and the like. In certain embodiments, EspA+Tircomprise at least 20% of the cell culture supernatant protein. Thecompositions can comprise cell culture supernatants and additionaladjuvants from more than one EHEC serotype to provide protection againstmultiple EHEC organisms. Moreover, a pharmaceutically acceptableadjuvant may be administered with the cell culture supernatant. Thecompositions are administered in an amount effective to elicit an immuneresponse to one or more of the secreted antigens, thereby reducing oreliminating EHEC infection. In some instances, EHEC colonization of theanimal is reduced or eliminated. In preferred embodiments, the animal isa cow or a sheep or other ruminant. In particularly preferredembodiments, the cell culture supernatant is derived from a cell cultureof EHEC O157:H7 or EHEC O157:NM.

[0074] Immunization with CCS stimulates the immune system of theimmunized animal to produce antibodies against one or more secreted EHECantigens, such as EspA, EspB, EspD and Tir, that block EHEC attachmentto intestinal epithelial cells, interfere with EHEC colonization and,thereby, reduce EHEC shedding by the animal. This reduction in EHECshedding results in a reduction in EHEC contamination of food and waterand a reduction in EHEC-caused disease in humans. Moreover, theunexpected and surprising ability of CCS immunization to prevent, reduceand eliminate EHEC colonization and shedding by cattle addresses along-felt unfulfilled need in the medical arts, and provides animportant benefit for humans.

[0075] Additionally, the CCS of the present invention can be used totreat or prevent EHEC infections in other mammals such as humans. Ifused in humans, the CCS can be produced from a mutated EHEC which hasbeen engineered to knock out one or both of the Shiga toxins 1 and 2 inorder to reduce toxicity.

[0076] As explained above, the therapeutic effectiveness of CCS can beincreased by adding thereto one or more of the secreted antigens inrecombinant or purified form, such as by adding recombinant or purifiedEspA, EspB, EspD, Tir, and the like, fragments thereof and/or analogsthereof. Intimin may also be added. Other methods to increase thetherapeutic effectiveness of CCS include, but are not limited to,complexing the CCS to natural or synthetic carriers and administeringthe CCS, before, at the same time as, or after another anti-EHEC agent.Such agents include, but are not limited to, biological, biologicallyengineered, chemical, nucleic acid based and recombinant proteinanti-EHEC agents.

[0077] CCS from pathogenic bacteria, other than serotypes of EHEC, thatrequire proteins such as EspA and Tir to colonize a host, can also beused to stimulate the immune system of an animal to produce antibodiesagainst secreted EHEC antigens that reduce bacterial binding tointestinal epithelial cells of the animal. These bacterial speciesinclude, but are not limited to Citrotobacter rodentium.

[0078] The CCS for use herein may be obtained from cultures of any EHECserotype, including, without limitation, EHEC serotypes from serogroupsO157, O158, O5, O8, O18, O26, O45, O48, O52, O55, O75, O76, O78,O84,O91, O103, O104, O111, O113, O114, O116, O118, O119, O121, O125,O28, O145, O146, O163, O165. Such EHEC serotypes are readily obtainedfrom sera of infected animals. Methods for isolated EHEC are well knownin the art. See, e.g., Elder et al., Proc. Natl. Acad. Sci. USA (2000)97:2999; Van Donkersgoed et al., Can. Vet. J. (1999) 40:332; VanDonkersgoed et al., Can. Vet. J. (2001) 42:714. Generally, such methodsentail direct plating on sorbitol MacConkey agar supplemented withcefixime and tellurite or immunomagnetic enrichment followed by platingon the same media. Moreover, CCS may be obtained from EHEC serotypesthat have been genetically engineered to knock-out expression of Shigatoxins 1 and/or 2, in order to reduce toxicity.

[0079] Generally, CCS is produced by culturing EHEC bacteria in asuitable medium, under conditions that favor type III antigen secretion.Suitable media and conditions for culturing EHEC bacteria are known inthe art and described in e.g., U.S. Pat. Nos. 6,136,554 and 6,165,743(incorporated herein by reference in their entireties), as well as in Liet al., Infec. Immun. (2000) 68:5090-5095; Fey et al., Emerg. Infect.Dis. (2000) Volume 6. A particularly preferable method of obtaining CCSis by first growing organisms in Luria-Bertani (LB) medium for a periodof about 8 to 48 hours, preferably about 12 to 24 hours, and dilutingthis culture about 1:5 to 1:50, preferably 1:5 to 1:25, more preferablyabout 1:10, into M-9 minimal medium supplemented with 20-100 mM NaHCO₂,preferably 30-50 mM, most preferably about 44 mM NaHCO₂, 4-20 mM MgSO₄,preferably 5-10 mM and most preferably about 8 mM MgSO₄, 0.1 to 1.5%glucose, preferable 0.2 to 1%, most preferably 0.4% glucose and 0.05 to0.5% Casamino Acids, preferably 0.07 to 0.2%, most preferably about 0.1%Casamino Acids. Cultures are generally maintained at about 37 degrees C.in 2-10% CO₂, preferably about 5% CO₂, to an optical density of about600 nm of 0.7 to 0.8. Whole cells are then removed by centrifugation andthe supernatant can be concentrated, e.g., 10-1000 fold or more, such as100-fold, using dialysis, ultrafiltration and the like. Total protein iseasily determined using methods well known in the art.

[0080] As explained above, the CCS can be supplemented with additionalEHEC secreted proteins, such as EspA, EspB, EspD and/or Tir. Intimin mayalso be added. These proteins can be produced recombinantly usingtechniques well known in the art. See, e.g., International PublicationNos. WO 97/40063 and WO 99/24576 for a description of the production ofrepresentative recombinant EHEC secreted proteins. In particular, thesequences for EspA, EspB, EspD, Tir and Intimin from various serotypesare known and described. See, e.g., GenBank Accession Nos. AE005594,AE005595, AP002566, AE005174, NC 002695, NC 002655 for the completesequence of the E. coli O157:H7 genome, which includes the sequences ofthe various O157:H7 secreted proteins. See, e.g., InternationalPublication No. WO 97/40063, as well as GenBank Accession Nos. Y13068,U80908, U5681, Z54352, AJ225021, AJ225020, AJ225019, AJ225018, AJ225017,AJ225016, AJ225015, AF022236 and AF200363 for the nucleotide and aminoacid sequences of EspA from a number of E. coli serotypes. See, e.g.,International Publication No. WO 99/24576, as well as GenBank AccessionNos. AF125993, AF132728, AF045568, AF022236, AF70067, AF070068,AF013122, AF200363, AF113597, AF070069, AB036053, AB026719, U5904 andU59502, for the nucleotide and amino acid sequences of Tir from a numberof E. coli serotypes. See, e.g., GenBank Accession Nos. U32312, U38618,U59503, U66102, AF081183, AF081182, AF130315, AF339751, AJ308551,AF301015, AF329681, AF319597, AJ275089-AJ275113 for the nucleotide andamino acid sequences of Intimin from a number of E. coli serotypes. See,e.g., GenBank Accession Nos. U80796, U65681, Y13068, Y13859, X96953,X99670, X96953, Z21555, AF254454, AF254455, AF254456, AF254457,AF054421, AF059713, AF144008, AF144009 for the nucleotide and amino acidsequences of EspB from a number of E. coli serotypes. See, e.g., GenBankAccession Nos. Y13068, Y13859, Y17875, Y17874, Y09228, U65681, AF054421and AF064683, for the nucleotide and amino acid sequences of EspD from anumber of E. coli serotypes.

[0081] These sequences can be used to design oligonucleotide probes andused to screen genomic or cDNA libraries for genes from other E. coliserotypes. The basic strategies for preparing oligonucleotide probes andDNA libraries, as well as their screening by nucleic acid hybridization,are well known to those of ordinary skill in the art. See, e.g., DNACloning: Vol. 1, supra; Nucleic Acid Hybridization, supra;Oligonucleotide Synthesis, supra; Sambrook et al., supra. Once a clonefrom the screened library has been identified by positive hybridization,it can be confirmed by restriction enzyme analysis and DNA sequencingthat the particular library insert contains a type III gene or a homologthereof. The genes can then be further isolated using standardtechniques and, if desired, PCR approaches or restriction enzymesemployed to delete portions of the full-length sequence.

[0082] Similarly, genes can be isolated directly from bacteria usingknown techniques, such as phenol extraction and the sequence furthermanipulated to produce any desired alterations. See, e.g., Sambrook etal., supra, for a description of techniques used to obtain and isolateDNA. Alternatively, DNA sequences encoding the proteins of interest canbe prepared synthetically rather than cloned. The DNA sequences can bedesigned with the appropriate codons for the particular amino acidsequence. In general, one will select preferred codons for the intendedhost if the sequence will be used for expression. The complete sequenceis assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge(1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay etal. (1984) J. Biol. Chem. 259:6311.

[0083] Once coding sequences for the desired proteins have been preparedor isolated, they can be cloned into any suitable vector or replicon.Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV 14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp 19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra; B.Perbal, supra.

[0084] The gene can be placed under the control of a promoter, ribosomebinding site (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired protein is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. Leader sequences can be removed by the hostin post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;4,425,437; 4,338,397.

[0085] Other regulatory sequences may also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Regulatory sequences are known to those of skill inthe art, and examples include those which cause the expression of a geneto be turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound. Other types ofregulatory elements may also be present in the vector, for example,enhancer sequences.

[0086] The control sequences and other regulatory sequences may beligated to the coding sequence prior to insertion into a vector, such asthe cloning vectors described above. Alternatively, the coding sequencecan be cloned directly into an expression vector which already containsthe control sequences and an appropriate restriction site.

[0087] In some cases it may be necessary to modify the coding sequenceso that it may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. It may also bedesirable to produce mutants or analogs of the protein. Mutants oranalogs may be prepared by the deletion of a portion of the sequenceencoding the protein, by insertion of a sequence, and/or by substitutionof one or more nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are describedin, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic AcidHybridization, supra.

[0088] The expression vector is then used to transform an appropriatehost cell. A number of mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Similarly,bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcusspp., will find use with the present expression constructs. Yeast hostsuseful in the present invention include inter alia, Saccharomycescerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha,Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii,Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.Insect cells for use with baculovirus expression vectors include, interalia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0089] Depending on the expression system and host selected, theproteins of the present invention are produced by culturing host cellstransformed by an expression vector described above under conditionswhereby the protein of interest is expressed. The protein is thenisolated from the host cells and purified. The selection of theappropriate growth conditions and recovery methods are within the skillof the art.

[0090] The proteins of the present invention may also be produced bychemical synthesis such as solid phase peptide synthesis, using knownamino acid sequences or amino acid sequences derived from the DNAsequence of the genes of interest. Such methods are known to thoseskilled in the art. See, e.g., J. M. Stewart and J. D. Young, SolidPhase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill.(1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis,Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, AcademicPress, New York, (1980), pp. 3-254, for solid phase peptide synthesistechniques; and M. Bodansky, Principles of Peptide Synthesis,Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., ThePeptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classicalsolution synthesis. Chemical synthesis of peptides may be preferable ifa small fragment of the antigen in question is capable of raising animmunological response in the subject of interest.

[0091] Once the above cell culture supernatants and, if desired,additional recombinant and/or purified proteins are produced, they areformulated into compositions for delivery to a mammalian subject. TheCCS is administered alone, or mixed with a pharmaceutically acceptablevehicle or excipient. Suitable vehicles are, for example, water, saline,dextrose, glycerol, ethanol, or the like, and combinations thereof. Inaddition, the vehicle may contain minor amounts of auxiliary substancessuch as wetting or emulsifying agents, pH buffering agents, or adjuvantsin the case of vaccine compositions, which enhance the effectiveness ofthe vaccine. Suitable adjuvants are described further below. Thecompositions of the present invention can also include ancillarysubstances, such as pharmacological agents, cytokines, or otherbiological response modifiers.

[0092] As explained above, vaccine compositions of the present inventionmay include adjuvants to further increase the immunogenicity of one ormore of the EHEC antigens. Such adjuvants include any compound orcompounds that act to increase an immune response to an EHEC antigen orcombination of antigens, thus reducing the quantity of antigen necessaryin the vaccine, and/or the frequency of injection necessary in order togenerate an adequate immune response. Adjuvants may include for example,emulsifiers, muramyl dipeptides, avridine, aqueous adjuvants such asaluminum hydroxide, chitosan-based adjuvants, and any of the varioussaponins, oils, and other substances known in the art, such as Amphigen,LPS, bacterial cell wall extracts, bacterial DNA, syntheticoligonucleotides and combinations thereof (Schijns et al., Curr. Opi.Immunol. (2000) 12:456), Mycobacterial phlei (M. phlei) cell wallextract (MCWE) (U.S. Pat. No. 4,744,984), M. phlei DNA (M-DNA), M-DNA-M.phlei cell wall complex (MCC). For example, compounds which may serve asemulsifiers herein include natural and synthetic emulsifying agents, aswell as anionic, cationic and nonionic compounds. Among the syntheticcompounds, anionic emulsifying agents include, for example, thepotassium, sodium and ammonium salts of lauric and oleic acid, thecalcium, magnesium and aluminum salts of fatty acids (i.e., metallicsoaps), and organic sulfonates such as sodium lauryl sulfate. Syntheticcationic agents include, for example, cetyltrimethylammonium bromide,while synthetic nonionic agents are exemplified by glyceryl esters(e.g., glyceryl monostearate), polyoxyethylene glycol esters and ethers,and the sorbitan fatty acid esters (e.g., sorbitan monopalmitate) andtheir polyoxyethylene derivatives (e.g., polyoxyethylene sorbitanmonopalmitate). Natural emulsifying agents include acacia, gelatin,lecithin and cholesterol.

[0093] Other suitable adjuvants can be formed with an oil component,such as a single oil, a mixture of oils, a water-in-oil emulsion, or anoil-in-water emulsion. The oil may be a mineral oil, a vegetable oil, oran animal oil. Mineral oil, or oil-in-water emulsions in which the oilcomponent is mineral oil are preferred. In this regard, a “mineral oil”is defined herein as a mixture of liquid hydrocarbons obtained frompetrolatum via a distillation technique; the term is synonymous with“liquid paraffin,” “liquid petrolatum” and “white mineral oil.” The termis also intended to include “light mineral oil,” i.e., an oil which issimilarly obtained by distillation of petrolatum, but which has aslightly lower specific gravity than white mineral oil. See, e.g.,Remington's Pharmaceutical Sciences, supra. A particularly preferred oilcomponent is the oil-in-water emulsion sold under the trade name ofEMULSIGEN PLUS™ (comprising a light mineral oil as well as 0.05%formalin, and 30 mcg/mL gentamicin as preservatives), available from MVPLaboratories, Ralston, Nebr. Suitable animal oils include, for example,cod liver oil, halibut oil, menhaden oil, orange roughy oil and sharkliver oil, all of which are available commercially. Suitable vegetableoils, include, without limitation, canola oil, almond oil, cottonseedoil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybeanoil, and the like.

[0094] Alternatively, a number of aliphatic nitrogenous bases can beused as adjuvants with the vaccine formulations. For example, knownimmunologic adjuvants include amines, quaternary ammonium compounds,guanidines, benzamidines and thiouroniums (Gall, D. (1966) Immunology11:369-386). Specific compounds include dimethyldioctadecylammoniumbromide (DDA) (available from Kodak) andN,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediarnine (“avridine”). Theuse of DDA as an immunologic adjuvant has been described; see, e.g., theKodak Laboratory Chemicals Bulletin 56(1):1-5 (1986); Adv. Drug Deliv.Rev. 5(3):163-187 (1990); J. Controlled Release 7:123-132 (1988); Clin.Exp. Immunol. 78(2):256-262 (1989); J. Immunol. Methods 97(2):159-164(1987); Immunology 58(2):245-250 (1986); and Int. Arch. Allergy Appl.Immunol. 68(3):201-208 (1982). Avridine is also a well-known adjuvant.See, e.g., U.S. Pat. No. 4,310,550 to Wolff, III et al., which describesthe use of N,N-higher alkyl-N′,N′-bis(2-hydroxyethyl)propane diamines ingeneral, and avridine in particular, as vaccine adjuvants. U.S. Pat. No.5,151,267 to Babiuk, and Babiuk et al. (1986) Virology 159:57-66, alsorelate to the use of avridine as a vaccine adjuvant.

[0095] Particularly preferred for use herein is an adjuvant known as“VSA3” which is a modified form of the EMULSIGEN PLUS™ adjuvant whichincludes DDA (see, U.S. Pat. No. 5,951,988, incorporated herein byreference in its entirety).

[0096] CCS vaccine compositions can be prepared by uniformly andintimately bringing into association the CCS preparations and theadjuvant using techniques well known to those skilled in the artincluding, but not limited to, mixing, sonication and microfluidation.The adjuvant will preferably comprise about 10 to 50% (v/v) of thevaccine, more preferably about 20 to 40% (v/v) and most preferably about20 to 30% or 35% (v/v), or any integer within these ranges.

[0097] The compositions of the present invention are normally preparedas injectables, either as liquid solutions or suspensions, or as solidforms which are suitable for solution or suspension in liquid vehiclesprior to injection. The preparation may also be prepared in solid form,emulsified or the active ingredient encapsulated in liposome vehicles orother particulate carriers used for sustained delivery. For example, thevaccine may be in the form of an oil emulsion, water in oil emulsion,water-in-oil-in-water emulsion, site-specific emulsion, long-residenceemulsion, sticky-emulsion, microemulsion, nanoemulsion, liposome,microparticle, microsphere, nanosphere, nanoparticle and various naturalor synthetic polymers, such as nonresorbable impermeable polymers suchas ethylenevinyl acetate copolymers and Hytrel® copolymers, swellablepolymers such as hydrogels, or resorbable polymers such as collagen andcertain polyacids or polyesters such as those used to make resorbablesutures, that allow for sustained release of the vaccine.

[0098] Furthermore, the polypeptides may be formulated into compositionsin either neutral or salt forms. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe active polypeptides) and which are formed with inorganic acids suchas, for example, hydrochloric or phosphoric acids, or organic acids suchas acetic, oxalic, tartaric, mandelic, and the like. Salts formed fromfree carboxyl groups may also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0099] Actual methods of preparing such dosage forms are known, or willbe apparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18thedition, 1990.

[0100] The composition is formulated to contain an effective amount ofsecreted EHEC antigen, the exact amount being readily determined by oneskilled in the art, wherein the amount depends on the animal to betreated and the capacity of the animal's immune system to synthesizeantibodies. The composition or formulation to be administered willcontain a quantity of one or more secreted EHEC antigens adequate toachieve the desired state in the subject being treated. For purposes ofthe present invention, a therapeutically effective amount of a vaccinecomprising CCS with or without added recombinant and/or purifiedsecreted EHEC antigens, contains about 0.05 to 1500 μg secreted EHECprotein, preferably about 10 to 1000 μg secreted EHEC protein, morepreferably about 30 to 500 μg and most preferably about 40 to 300 μg, orany integer between these values. EspA+Tir, as well as other EHECantigens, may comprise about 10% to 50% of total CCS protein, such asabout 15% to 40% and most preferably about 15% to 25%. If supplementedwith rEspA+rTir, the vaccine may contain about 5 to 500 μg of protein,more preferably about 10 to 250 μg and most preferably about 20 to 125μg.

[0101] Routes of administration include, but are not limited to, oral,topical, subcutaneous, intramuscular, intravenous, subcutaneous,intradermal, transdermal and subdermal. Depending on the route ofadministration, the volume per dose is preferably about 0.001 to 10 ml,more preferably about 0.01 to 5 ml, and most preferably about 0.1 to 3ml. Vaccine can be administered in a single dose treatment or inmultiple dose treatments (boosts) on a schedule and over a time periodappropriate to the age, weight and condition of the subject, theparticular vaccine formulation used, and the route of administration.

[0102] Any suitable pharmaceutical delivery means may be employed todeliver the compositions to the vertebrate subject. For example,conventional needle syringes, spring or compressed gas (air) injectors(U.S. Pat. No. 1,605,763 to Smoot; U.S. Pat. No. 3,788,315 to Laurens;U.S. Pat. No. 3,853,125 to Clark et al.; U.S. Pat. No. 4,596,556 toMorrow et al.; and U.S. Pat. No. 5,062,830 to Dunlap), liquid jetinjectors (U.S. Pat. No. 2,754,818 to Scherer; U.S. Pat. No. 3,330,276to Gordon; and U.S. Pat. No. 4,518,385 to Lindmayer et al.), andparticle injectors (U.S. Pat. No. 5,149,655 to McCabe et al. and U.S.Pat. No. 5,204,253 to Sanford et al.) are all appropriate for deliveryof the compositions.

[0103] If a jet injector is used, a single jet of the liquid vaccinecomposition is ejected under high pressure and velocity, e.g., 1200-1400PSI, thereby creating an opening in the skin and penetrating to depthssuitable for immunization.

[0104] The following examples will serve to further illustrate thepresent invention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

[0105] C. Experimental

EXAMPLE 1

[0106] Preparation of Cell Culture Supernatant (CCS)

[0107] Wild type EHEC O157:H7 were grown under conditions to maximizethe synthesis of CCS proteins (Li et al., Infect. Immun. (2000)68:5090). Briefly, an overnight standing culture of EHEC O157:H7 wasgrown in Luria-Bertani (LB) medium overnight at 37° C. (±5% CO₂). Theculture was diluted 1:10 in M-9 minimal medium supplemented with 0.1%Casamino Acids, 0.4% glucose, 8 mM MgSO₄ and 44 mM NaHCO₂. Cultures weregrown standing at 37° C. in 5% CO₂ to an optical density at 600 nm of0.7 to 0.8 (6-8 h). Bacteria were removed by centrifugation at 8000 rpmfor 20 min at 4° C. The supernatant was concentrated 100 fold byultrafiltration and total protein was determined by the bicinchoninicacid protein assay method.

[0108]FIG. 1 shows molecular weight markers (lane 1) and a typical CCSprotein profile obtained by electrophoresis of CCS in a SDS-10%polyacrylamide gel (SDS-PAGE) followed by Coomassie blue staining (lane2). The positions of EspA (25 kD), EspB/EspD (40 kD), undegraded Tir (70kD) and degraded Tir (55 kD) are indicated. As determined bydensitometric analysis using an HP Scanjet 5100C and the ID softwareprogram from Advance American Biotechnology (Fullerton, Calif., USA),EspA was about 5% undegraded Tir about 20% and degraded Tir about 6% ofthe total protein. However, the percentages of proteins determined bydensitometric analysis of Coomassie blue stained SDS-polyacrylamide gelsis not exact due to variations in background staining, variations in theuptake of the Coomassie blue stain, variations in the density of thebands, and other factors known to those skilled in the art.

EXAMPLE 2

[0109] Preparation of Recombinant Proteins

[0110] The genes coding for EspA, EspB, Intimin and Tir were isolated(Li et al., Infect. Immun. (2000) 68:5090). A clinical isolate of EHECO157:H7 was used as the source of DNA. EspA, EspB, Tir, and the regionof eae encoding the 280 carboxyl-terminal amino acids of Intimin wereamplified from chromosomal DNA using PCR to introduce unique restrictionsites, followed by cloning into appropriate plasmids. The resultingplasmids were cleaved and ligated to create histidine-tagged fusions.Plasmids were electrocuted into an expression strain of E. coli and theE. coli were propagated (Ngeleka et al., Infect. Immun. (1996) 64:3118).Gene expression was driven using the Tac promoter following IPTG(isopropyl-β-D-thiogalactopyranoside) induction. Bacteria were pelleted,resuspended in Tris-buffered saline and lysed by sonication. The lysatewas centrifuged to remove insoluble material and the histidine-taggedproteins were purified by passage through a solid-phase nickel affinitychromatography column that specifically binds proteins containing thehistidine tag. All recombinant protein preparations were stored at −20°C. until use.

[0111] The purity of the recombinant proteins was assessed by SDS-PAGEon 10% gels followed by Coomassie blue staining. Typical gel profiles ofthe chromatographically purified recombinant (r) proteins are shown inFIG. 2. rEspA (lane 2) rEspB (lane 3) and rIntimin (lane 4), wererecovered in relatively pure form, but rTir (lane 5) was subject to somedegradation.

EXAMPLE 3

[0112] Vaccine Formulation and Delivery

[0113] Vaccines were formulated by mixing CCS or rEspA+rTir in 2 ml of acarrier containing from 30 to 40% of an adjuvant. Vaccines weredelivered subcutaneously. Animals were immunized on day 1 and again at a3-4 week intervals (boost). Serum samples were obtained prior to thefirst immunization, at the time of each boost and at the end of theexperiment.

[0114] The serological response to immunization was determined using anenzyme-linked inmunosorbent assay (ELISA). One hundred μl of rEspA (0.16μg/well), rTir (0.1 μg/well), rEspB (0.24 μg/well) and rIntimin (0.187μg/well) were used to coat the wells in microtiter plates and the plateswere incubated overnight at 4° C. The wells were washed 3×, blocked with0.5% nonfat dried milk in phosphate-buffered saline. Serial dilutions ofsera were added to each well and incubated for 2 h at 37° C. The wellswere washed and blocked and 100 μl of peroxidase-conjugated rabbitanti-bovine immunoglobulin G antibodies (1:5000) were added to each wellfor 1 h at 37° C. The wells were washed and plates were read at awavelength of 492 nm.

EXAMPLE 4

[0115] Experimental Animals

[0116] Cattle, between the ages of 8 and 12 months, were purchased fromlocal ranchers. Fecal samples were obtained daily from each animal for14 days. The number of EHEC O157:H7 in the fecal samples was determinedby plating on Rainbow Agar. The plates were incubated at 37° C. for 2days and black colonies were enumerated. Growth was scored from 0-5.Animals having a score of 0 (no EHEC O157:H7) were used in allexperiments.

EXAMPLE 5

[0117] Animal Colonization Model

[0118] A model for EHEC O157:H7 colonization of cattle, wherein theinfection was sustained for >2 months, was developed using adose-titration protocol.

[0119] EHEC O157:H7 were grown as in Example 1. Twenty-four cattle weredivided into 3 groups of 8 animals each. Group 1 received 10^(6,) Group2 10⁸ and Group 3 10¹⁰ CFU of EHEC O157:H7 by oral-gastric intubation ina volume of 50 ml on day 0.

[0120] To monitor shedding, fecal material was collected on days 1through 14. The fecal material was weighed, suspended in sterile salineand inoculated into culture media. Culture density was determined as inExample 1.

[0121] As shown in FIG. 3, there was no significant difference betweennumbers of EHEC O157:H7 shed by Group 2 (10⁸ CFU) and Group 3 (10¹⁰ CFU)cattle. Group 2 cattle shed the most EHEC O157:H7 on each of the 14days. The number of EHEC O157,.H7 shed by Group 2 cattle reached amaximum on day 6 and declined to zero by day 14.

[0122] Animals shedding EHEC O157:H7 (hereinafter, “positive”) were keptan additional 40 days during which time the number of EHEC O157:H7 sheddecreased to an undetectable level. The shedding of EHEC O157:H7 bypreviously positive animals (hereinafter, “carriers”) was reactivated bywithholding feed for 24 hours and vaccinating withcommercially-available clostridial or H. somnus vaccines. As shown inFIG. 4, the number of carrier animals shedding EHEC O157:H7 reached amaximum of approximately 50% on days 6 and 7 post-reactivation anddeclined to zero by day 15.

[0123] As a dose of 10⁸ CFU produced a detectable number of shed EHECO157:H7 during the 14 days post-infection (FIG. 3) and resulted inpersistently infected animals (FIG. 4), this dose was used as thechallenge dose in subsequent experiments.

EXAMPLE 6

[0124] Protective Capacity of CCS

[0125] To test the vaccine potential of secreted proteins, CCS was mixedwith the oil-based adjuvant, VSA3 (U.S. Pat. No. 5,951,988, incorporatedherein by reference in its entirety; S. van Drunen Littel-van den Hurket al., Vaccine (1993) 11:25) such that each 2 ml dose contained 200 μgof CCS protein and 30% (v/v) of adjuvant (CCS vaccine). For the controlgroup, sterile saline was mixed with VSA3, such that each 2 ml dosecontained 0 μg of CCS protein and 30% (v/v) of adjuvant (salinevaccine).

[0126] Sixteen cattle were divided in 2 groups of eight animals each.Group 1 cattle received 2 ml of CCS vaccine subcutaneously(experimental) and Group 2 cattle received 2 ml saline vaccinesubcutaneously (control) on days 1 and 22 (boost). Seroconversion wasassayed by ELISA (Example 3), on days 1 (pre-immunization), 22 and 36.As shown in Table 1, at day 22, Group 1 animals showed specific antibodytiters to EspA and Tir and, at day 36, these titers showed a significantincrease. Group 2 animals showed no specific antibody titers at days 22and 36. In particular, the group which received the EHEC vaccine showeda 13-fold increase in specific antibody titer to type III secretedproteins after a single immunization and following the first booster,the eight animals in the EHEC vaccine group demonstrated a 45-foldincrease in specific antibody titer while only one of the placebovaccine group seroconverted (X², p=0.0002). TABLE 1 Serological responseto immunization with CCS Specific Antibody Titers*-Group MeansPre-immunization Boost Challenge Group (Day 1) (Day 22) (Day 36) 1.Experimental 350 5,000 12,500 2. Control 450   500   650

[0127] At day 36, Group 1 and Group 2 animals were challenged with 10⁸CFU of EHEC O157:H7 by oral-gastric intubation and fecal shedding wasmonitored for 14 days (Example 5). As summarized in Table 2, fewerexperimental animals shed EHEC O157:H7 than control animals andexperimental animals that did shed, shed EHEC O157:H7 for a shorterperiod of time than control animals (FIG. 7). In particular, The mediannumber of days during which the organism was shed in the vaccinatedanimals was 1.5 compared to 3.5 in the placebo group (Wilcoxin SignedRank Test, p=0.08). Seven out of eight placebo-immunized animals shedthe bacteria during the trial and four of those animals shed thebacteria for four or more consecutive days, indicating that they werepersistently infected. Five out of eight EHEC vaccine-immunized animalsshed bacteria at some point during the trial but only one animal shedthe organism for more than two consecutive days, indicating thatcolonization was transient and significantly less than the placebogroup. The total number of bacteria isolated from fecal samples wassignificantly lower among the EHEC-vaccinated group as compared to theplacebo group (Wilcoxin Signed Rank Test, p=0.05), with the formerhaving a median of 6.25 colony forming units (CFU) per gram of fecesrecovered compared to a median value of 81.25 CFU/g for the latter.Thus, vaccination with the type III-secreted proteins appeared to reducethe ability of the organism to colonize the intestine as reflected bythe decrease in the number of days animals shed the organism as well asthe numbers of shed bacteria detected by fecal culture. TABLE 2 Sheddingby experimental and control animals Experimental Control Animalsshedding >1 day 1/8 6/8 Number of days with scores of >1 1 8 Averagedays of shedding per animal 0.875 2.5 Total days shedding per group 7 20

[0128] These data show that CCS induced an antibody response in cattlethat reduced both number of animals shedding EHEC O157:H7 and the numberof days during which EHEC O157:H7 were shed.

[0129] In order to enhance the effectiveness of the vaccine formulation,groups of 6 calves were immunized as described above with one of threedoses of secreted proteins (50 μg, 100 μg, 200 μg) or a placebo and theserological response was measured in serum samples taken at days 0, 21(boost) and 35. No significant difference in anti-EHEC, anti-Tir oranti-EspA responses were observed between any of the groups whichreceived the EHEC vaccine at any time point but all three weresignificantly higher than the placebo group on days 21 and 35. Thus, asecond vaccine trial was designed in which three groups of yearlingcattle were immunized three times with 50 μg of secreted proteins(n=13), 50 μg of secreted proteins from a tir mutant (ΔTir, n=10) or aplacebo (n=25). The adjuvant used was VSA3 and animals were immunized bysubcutaneous injection on days 0, 21, and 35, followed by oral challengewith E. coli O157:H7 on day 49. The serological response to immunizationis shown in Table 3 (days 0 and 49 only) and was comparable to thatobserved in the trial described above. The group which received the ΔTirvaccine showed a response of similar magnitude against total secretedproteins as the group which received the vaccine prepared from thewild-type strain, but, as expected, a significantly reduced response toTir (Wilcoxin Signed Rank Test, p=0.006). However, the former group didshow an increase in anti-Tir antibody levels (Wilcoxin Signed Rank Test,p=0.009), indicating either exposure to an organism producing animmunologically related molecule or natural exposure to E. coli O157:H7.This is further supported by the observation that there was asignificant increase in the anti-Tir antibody titer in the placebo groupon the day of challenge (Wilcoxin Signed Rank Test, p=0.002) but nodifference between the placebo or ΔTir groups (p=0.37, Kruskal-WallisANOVA). The response to EspA was similar in both the EHEC and ΔTirvaccine groups (p=0.45, Kruskal-Wallis ANOVA) and was significantlyhigher than the placebo-immunized animals (p<0.0001). TABLE 3 Medianserological response to immunization with secreted proteins preparedfrom wild- type E. coli O157:H7 (EHEC), an isogenic tir mutant (ΔTir) ora placebo. Titers are expressed as geometric mean values of the lastpositive dilution of sera ( ). Numbers in parentheses represent the25^(th)-75^(th) percentile. Anti-EHEC Anti-Tir Anti-EspA Group n Day 0Day 49 Day 0 Day 49 Day 0 Day 49 EHEC 13 10 6400 100 1600 100 400(10-100) (3200-12800) (10-200) (800-3200) (10-200) (200-1600) ΔTir 10 106400 10 200 100 300 (10-100) (3200-25600) (10-200) (100-800) (10-200)(100-1600) Placebo 25 10 10 100 200 100 100 (10-200) (10-200) (10-200)(10-400) (10-200) (10-200)

[0130] The immune response against each vaccine formulation was alsoanalyzed qualitatively by Western blotting using sera from tworepresentative animals per group. The results for representative animalsare shown in FIG. 8 and demonstrate that the proteins secreted by thetype III system were highly immunogenic in cattle. The response in theEHEC and ΔTir vaccine groups was similar with the exception of theresponse against Tir which was absent in the latter group (FIG. 8, toppanels). EspB, EspD and Tir were all reactive, and following the secondimmunization on day 21 a significant response against lipopolysaccharidewas also observed. The kinetics of the immune response in a vaccinatedanimal (FIG. 8, bottom panels) show that anti-Tir antibodies weredetectable following a single immunization, as were antibodies against43-kDa and 100-kDa proteins. The latter proteins were produced by thewild-type strain as well as the sepB and tir mutants and the 100 kDaprotein is probably EspP, a non-type III EHEC secreted protein.

[0131] Following oral challenge with E. coli O157:H7 on day 49, eachgroup was monitored daily for fecal shedding of the organism for 14days. In this experiment, bacteria were cultured followingimmunomagnetic enrichment (J. Van Donkersgoed et al., Can. Vet. J.(2001) 42:714, Chapman and Siddons, J. Med. Microbiol. (1996) 44:267)rather than direct plating since yearling cattle shed less than calvesin this infection model. On the day of challenge, two animals in theplacebo group were culture-positive for E. coli O157:H7 and wereeliminated from the trial. The placebo-immunized animals shed theorganism after challenge much more than those in the two EHEC vaccinegroups (FIG. 9). Those which received the placebo vaccine shed theorganism for a median of 4 days, significantly longer than the median of0 days by the other two vaccine groups (p=0.0002, Kruskal-Wallis ANOVA).Significantly fewer bacteria were recovered from the EHEC and ΔTirvaccine groups (p=0.04, Kruskal-Wallis ANOVA). From day 2 post-infectiononwards, 78% of the placebo animals shed the organism for at least oneday as compared to 15% of the EHEC and 30% of the ΔTir vaccinates (Table4).

[0132] The data presented above demonstrate that virulence factors ofEHEC, namely those secreted by the type III system, can be used aseffective vaccine components for the reduction of colonization of cattleby EHEC bacteria, such as EHEC O157:H7. These proteins are major targetsof the immune response in humans following infection (Li et al., Infect.Immun. (2000) 68:5090), although cattle do not usually mount asignificant serological response against these proteins followingnatural exposure to the organism. However, animals vaccinated with theseproteins are primed and show an increase in anti-EHEC and anti-Tirtiters following oral challenge with the organism.

[0133] Tir is likely required for colonization of the bovine intestine,and this is supported by the observation that a vaccine containingsecreted proteins from a ΔTir E. coli O157:H7 strain was not asefficacious as an identical formulation from an isogenic wild-typeisolate. However, the former vaccine was significantly more efficaciousthan a placebo suggesting that immunity against colonization ismultifactorial in nature. This is supported by the Western blot analysisof the response to immunization in which several protein components aswell as lipopolysaccharide were recognized. The contribution toprotection by lipopolysaccharide is not known, but the presence ofantibodies against this molecule does not correlate with protection in amurine EHEC model (Conlan et al., Can. J. Microbiol. (1999) 45:279;Conlan et al., Can. J. Microbiol. (2000) 46:283). Also, immunizationwith recombinant Tir and EspA can reduce numbers of bacteria shed, butnot the actual numbers of animals nor the duration of shedding.

[0134] The prevalence of non-O157 serotypes in North America appears tobe increasing and represents a significant portion of EHEC infections inother geographical locations. Since the type III-secreted antigensappear to be relatively conserved among non-O157 EHEC serotypes, thisvaccine formulation is likely broadly cross-protective, in contrast toformulations based upon the O157 LPS antigen. TABLE 4 Number of animalsshedding E. coli O157:H7 at any time between day 2 and day 14post-challenge. Number Percent Vaccine Shedding n Shedding p-value EHEC2 13 15.4 0.003 ΔTir 3 10 30   0.008 Placebo 18  23 78.3 1   

EXAMPLE 7

[0135] Protective Capacity of rEspA+rTir and rEspB+rIntimin

[0136] rEspA, rTir, rEspB and rIntimin were mixed with the oil-basedadjuvant, VSA3, such that each 2 ml dose contained 50 μg of rEspA+rTiror of rEspB+rIntimin and 30% (v/v) of adjuvant. Sterile saline was mixedwith VSA3, such that each 2 ml dose contained 0 μg of rEspA+rTir or ofrEspB+rIntimin and 30% (v/v) of adjuvant.

[0137] Thirty four cattle were divided in 4 groups. Ten cattle, Group 1,were immunized with rEspA+rTir vaccine (experimental) and 10 cattle,Group 2, were immunized with rEspB+rIntimin vaccine (experimental) ondays 1, 22 (boost) and 36. Seven cattle, Group 3, and 7 cattle, Group 4,were immunized with saline vaccine (control) an days 1, 22 (boost) and36. Seroconversion was assayed by ELISA (Example 3) on days 1(pre-immunization), 22 and 36. As shown in FIG. 5, at day 22, Group 1animals showed specific antibody titers to rEspA and to rTir and Group 2animals showed specific antibody titers to rEspB and to rIntimin. Also,as shown in FIG. 5, at day 36, Group 1 animals showed an increase inspecific antibody titer to rTir and no change in specific antibody titerto rEspA and Group 2 animals showed an increase in specific antibodytiter to rIntimin and a decrease in specific antibody titer to rEspB.Groups 3 and 4 animals showed no specific antibody titers at days 22 and36.

[0138] At day 36, Groups 1-4 animals were challenged with 10⁸ CFU ofEHEC O157:H7 and shedding was monitored daily for 14 days (Example 5).As shown in FIG. 6, differences in shedding between Group 1 (rTir+rEspA)animals and Group 3 (saline) animals was minimal during the first 5 dayspost-challenge. However, during the second week post-challengedifferences in Group 1 animals and Group 3 animals were evident. FewerGroup 1 animals shed EHEC O157:H7 than Group 3 animals. Group 1 animalsshed less EHEC O157:H7 in their feces for shorter time periods thanGroup 3 animals. Differences in shedding between Group 2(rEspB+rIntimin) and Group 4 (saline) animals were not evident withrespect to the number of animals shedding, the number of EHEC O157:H7shed and the time period of shedding.

[0139] These data show that the antibody response induced by rEspA+rTirvaccine interfered with EHEC O157:H7 colonization of cattle, whereas theantibody response induced by rEspB+rIntimin vaccine did not interferewith EHEC O157:H7 colonization of cattle.

EXAMPLE 8

[0140] Protective Capacity of CCS+rEspA+rTir

[0141] CCS, CCS+rEspA, CCS+rTir, CCS+rEspA+rTir and saline are mixedwith an adjuvant.

[0142] Twenty-five cattle are divided into 5 groups of five 5 cattle andare immunized an days 1 and 22 (boost). Group 1 receives CCS vaccine,Group 2 CCS+rEspA vaccine, Group 3 CCS+rTir vaccine, Group 4CCS+rEspA+rTir vaccine, and Group 5 saline vaccine. Seroconversion isassayed by ELISA (Example 3) on days 1 (pre-immunization), 22 (boost)and 36. On days 22 and 36 each of Groups 1-5 animals show specificantibody titers against EspA and Tir, whereas Group 6 animals show nospecific antibody titers.

[0143] At day 36, Groups 1-5 animals are challenged with 10⁸ CFU of EHECO157:H7 and shedding is monitored daily for 14 days (Example 5). Feweranimals in Groups 1-4 shed EHEC O157:H7 than animals in Group 5. Group 5animals shed the most EHEC O157:H7; Group 1 animals shed less EHECO157:H7 than Group 5 animals and Groups 2-4 animals shed less EHECO157:H7 than Group 1 animals.

EXAMPLE 9

[0144] Protective Capacity of CCS with Various Antigens

[0145] CSS is mixed with and adjuvant, such that each 2 ml dose contains0, 50, 100 or 200 μg of CCS and 30% (v/v) of adjuvant (Table 5). TABLE 5Protective capacity of CCS with various adjuvants Antigen Group μgAdjuvant CCS 1  50 Emulsigen-Plus CCS 2 100 Emulsigen-Plus CCS 3 200Emulsigen-Plus CCS 4 200 Carbigen CCS 5 100 MCC CCS 6 200 MCC CCS 7 200MCC + Carbigen CCS 8 200 VSA CCS 9  0 Emulsigen-Plus (control)

[0146] Seventy-two cattle are divided in 9 groups of 8 cattle. Groups1-8 animals are immunized with CCS+adjuvant (Table 5) and Group 9 cattleare immunized with saline+adjuvants on days 1 and 22 (boost).Seroconversion is assayed by ELISA (Example 3) on days 1(pre-immunization), 22 (boost) and 36. Groups 1-8 (CCS+adjuvant) animalsshow specific antibody titers to EspA and Tir on days 22 and 36. Group 9(saline+adjuvant) animals show no specific antibody titers on days 22and 36.

EXAMPLE 10

[0147] Protective Capacity of CCS in Dairy Cows

[0148] Twenty adult dairy cows are divided in 2 groups of 10 cows. Group1 is immunized with CCS vaccine and Group 2 is immunized withsaline-vaccine on days 1 and day 22 (boost). Seroconversion is assayedby ELISA (Example 3) on days 1 (pre-immunization), 22 and 36. On days 22and 36 Group 1 cows show specific antibody titers against EspA and Tir,whereas Group 2 cows show no specific antibody titers.

[0149] At day 36, Groups 1 and 2 cows are challenged with 10⁸ CFU ofEHEC O157:H7 and shedding is monitored daily for 14 days (Example 5).Fewer Group 1 cows shed EHEC O157:H7 than Groups 2 cows. Group 1 cowsshed less EHEC O157:H7 for a shorter period of time than Groups 2 cows.

[0150] Six months after the initial immunization, Group 1 and 2 cows areagain immunized (2nd boost) via the subcutaneous route. On day 14following the 2nd boost, antibody titers are assayed by ELISA (Example3). Group 1 cows have specific antibody titers to EspA and Tir, whereasGroup 2 cows have no specific antibody titers.

[0151] On day 14 following the 2nd boost, Groups 1 and 2 cows are againchallenged with 10⁸ CFU of EHEC O157:H7 and shedding is monitored dailyfor 14 days (Example 5). Fewer Group 1 (CCS) cows shed EHEC O157:H7 thanGroup 2 (saline) cows. Group 1 cows shed less EHEC O157:H7 for a shortertime periods than Group 2 cows.

EXAMPLE 11

[0152] Protective Capacity of CCS in Calves

[0153] Ten weaned calves (3-6 month old) are divided into 2 groups of 5calves and are immunized prior to entry into a feed-lot (day 0) and onthe day of entry into a feed lot (day 1, boost). Group 1 calves receiveCCS vaccine and Group 2 calves receive saline vaccine. Seroconversion isassayed by ELISA (Example 3) on days 0, 1 and 14. On days 1 and 14 Group1 (CCS) calves show specific antibody titers to EspA and Tir, whereasGroup 2 (saline) calves show no specific antibody titers.

[0154] At day 14, Groups 1 and 2 calves are challenged with 10⁸ CFU ofEHEC O157:H7 and shedding is assayed daily for 14 days (Example 5).Fewer Group 1 calves shed EHEC O157:H7 than Group 2 calves. Group 1calves shed less EHEC O157:H7 for a shorter time period than Group 2calves.

[0155] Ten weaned calves (3-6 mouth old) we divided into 2 groups of 5calves and are immunized on the day of entry into a feed-lot (day 1) andon day 22 (boost) in the feed lot. Group 1 calves receive CCS vaccineand Group 2 calves receive saline vaccine. Seroconversion is assayed byELISA (Example 3) on days 1 (pre-immunization), 22 and 36. On days 22and 36 Group 1 (CCS) calves show specific antibody titers to EspA andTir, whereas Group 2 (saline) calves show no specific antibody titers.

[0156] At day 36, Groups 1 and 2 calves are challenged with 10⁸ CFU ofEHEC O157:H7 and shedding is assayed daily for 14 days (Example 5).Fewer Group 1 calves shed EHEC O157:H7 than Group 2 calves. Group 1calves shed less EHEC O157:H7 for a shorter time period than Group 2calves.

EXAMPLE 12

[0157] Protective Capacity of CCS in Sheep

[0158] Twenty adult sheep are divided in 2 groups of 10 sheep. Group 1is immunized with CCS vaccine and Group 2 is immunized with salinevaccine on day 1 and day 22 (boost). Seroconversion is assayed by ELISA(Example 3) on days 1 (pre-immunization), 22 and 36. On days 22 and 36Group 1 sheep show specific antibody titers against EspA and Tir,whereas Group 2 sheep show no specific antibody titers.

[0159] At day 36, Groups 1 and 2 sheep are challenged with 10⁸ CFU ofEHEC O157:H7 and shedding is monitored daily for 14 days (Example 5).Fewer Group 1 sheep shed EHEC O157:H7 than Group 2 sheep. Group 1 sheepshed less EHEC O157:H7 for a shorter period of time than Group 2 sheep.

[0160] Thus, compositions and methods for treating and preventingenterohemorragic E. coli colonization of mammals have been disclosed.Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

We claim:
 1. A vaccine composition comprising an enterohemorragicEscherichia coli (EHEC) cell culture supernatant and an immunologicaladjuvant.
 2. The vaccine composition of claim 1, wherein the EHEC isEHEC O157:H7.
 3. The vaccine composition of claim 1, wherein the EHEC isEHEC O157:NM.
 4. The vaccine composition of claim 1, wherein theimmunological adjuvant comprises an oil-in-water emulsion.
 5. Thevaccine composition of claim 2, wherein the immunological adjuvantcomprises an oil-in-water emulsion.
 6. The vaccine composition of claim4, wherein the immunological adjuvant comprises a mineral oil anddimethyldioctadecylammonium bromide.
 7. The vaccine composition of claim5, wherein the immunological adjuvant comprises a mineral oil anddimethyldioctadecylammonium bromide.
 8. The vaccine composition of claim6, wherein the immunological adjuvant is VSA3.
 9. The vaccinecomposition of claim 7, wherein the immunological adjuvant is VSA3. 10.The vaccine composition of claim 8, wherein VSA3 is present in thecomposition at a concentration of about 20% to about 40% (v/v).
 11. Thevaccine composition of claim 10, wherein VSA3 is present in thecomposition at a concentration of about 30% (v/v).
 12. The vaccinecomposition of claim 9, wherein VSA3 is present in the composition at aconcentration of about 20% to about 40% (v/v).
 13. The vaccinecomposition of claim 12, wherein VSA3 is present in the composition at aconcentration of about 30% (v/v).
 14. The vaccine composition of claim1, further comprising one or more recombinant or purified EHEC antigensselected from the group consisting of EspA, EspB, EspD, Tir and Intimin.15. The vaccine composition of claim 14, wherein EspA+Tir comprise atleast 20% of the cell protein present in the composition.
 16. A methodfor eliciting an immunological response in a mammal against a secretedenterohemorragic Escherichia coli (EHEC) antigen, said method comprisingadministering to said mammal a therapeutically effective amount of acomposition comprising an EHEC cell culture supernatant.
 17. The methodof claim 16, wherein the EHEC is EHEC O157:H7.
 18. The method of claim16, wherein the mammal is a ruminant.
 19. The method of claim 18,wherein the ruminant is a bovine subject.
 20. The method of claim 16,wherein the composition further comprises an immunological adjuvant. 21.The method of claim 17, wherein the composition further comprises animmunological adjuvant.
 22. The method of claim 20, wherein theimmunological adjuvant comprises an oil-in-water emulsion.
 23. Themethod of claim 21, wherein the immunological adjuvant comprises anoil-in-water emulsion.
 24. The method of claim 22, wherein theimmunological adjuvant comprises a mineral oil anddimethyldioctadecylammonium bromide.
 25. The method of claim 23, whereinthe immunological adjuvant comprises a mineral oil anddimethyldioctadecylammonium bromide.
 26. The method of claim 24, whereinthe immunological adjuvant is VSA3.
 27. The method of claim 25, whereinthe immunological adjuvant is VSA3.
 28. The method of claim 16, whereinthe composition further comprises one or more recombinant or purifiedEHEC antigens selected from the group consisting of EspA, EspB, EspD,Tir and Intimin.
 29. The method of claim 28, wherein EspA+Tir compriseat least 20% of the cell protein present in the composition.
 30. Amethod for eliciting an immunological response in a ruminant against asecreted enterohemorragic Escherichia coli O157:H7 (EHEC O157:H7)antigen, said method comprising administering to said ruminant atherapeutically effective amount of a composition comprising an EHECO157:H7 cell culture supernatant and VSA3.
 31. The method of claim 30,wherein VSA3 is present in the composition at a concentration of about20% to about 40% (v/v).
 32. The method of claim 31, wherein VSA3 ispresent in the composition at a concentration of about 30% (v/v).
 33. Amethod for reducing colonization of enterohemorragic Escherichia coli(EHEC) in a ruminant comprising administering to said ruminant atherapeutically effective amount of a composition comprising an EHECcell culture supernatant and an immunological adjuvant.
 34. A method forreducing shedding of enterohemorragic Escherichia coli (EHEC) from aruminant comprising administering to said ruminant a therapeuticallyeffective amount of a composition comprising an EHEC cell culturesupernatant and an immunological adjuvant.